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The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages.

Mraheil MA, Billion A, Mohamed W, Mukherjee K, Kuenne C, Pischimarov J, Krawitz C, Retey J, Hartsch T, Chakraborty T, Hain T - Nucleic Acids Res. (2011)

Bottom Line: Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments.A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly.Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, 35392 Giessen, Germany.

ABSTRACT
Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500 nt) isolated from extracellularly growing bacteria and from L. monocytogenes infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here, we report on the discovery of 150 putative regulatory RNAs of which 71 have not been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50* for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNA candidates and the absence of sRNA loci in genomes of naturally occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

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Northern blots of sRNA candidates. Validation of RNA-seq data with northern blot analysis of extracellularly expressed sRNAs from bacteria at mid exponential growth phase in BHI (EC) compared with intracellularly expressed sRNAs at 4 h post-infection in murine macrophages (IC) of rli31, rli33-1, rli50 and rli112.
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Figure 2: Northern blots of sRNA candidates. Validation of RNA-seq data with northern blot analysis of extracellularly expressed sRNAs from bacteria at mid exponential growth phase in BHI (EC) compared with intracellularly expressed sRNAs at 4 h post-infection in murine macrophages (IC) of rli31, rli33-1, rli50 and rli112.

Mentions: The results of the northern blotting, shown in Figure 2, demonstrate signals corresponding to small transcripts from each of the candidates. According to the signals in the northern blot analysis, some of the sRNA candidates were differentially expressed under the tested conditions. rli31 and rli33-1 showed variation in transcript concentration during extra- and intra-cellular growth. Both sRNA candidates exhibited significantly higher transcript numbers during intracellular growth. The northern blot results correlate with read-numbers from transcriptome deep sequencing (Supplementary Table S3). The determined size of rli33-1 by northern blot analysis of ∼140 nt confirms the read length (144 nt) assessed by RNA-seq. rli33-1 showed two bands in the northern blot at ∼200 and 130 nt in length, that probably originate from processing of the larger co-transcript. Both fragments were also detected by transcriptome sequencing excluding the possibility of cross-hybridization (Figure 2). rli50 appears to be longer than predicted by sequencing (306 nt) and has a size of ∼350 nt as determined by northern blot. We observed with an rli112 specific probe strong northern blot signals (∼140 nt) under extra- and intra-cellular conditions indicating high RNA abundance of this new unpublished putative sRNA.Figure 2.


The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages.

Mraheil MA, Billion A, Mohamed W, Mukherjee K, Kuenne C, Pischimarov J, Krawitz C, Retey J, Hartsch T, Chakraborty T, Hain T - Nucleic Acids Res. (2011)

Northern blots of sRNA candidates. Validation of RNA-seq data with northern blot analysis of extracellularly expressed sRNAs from bacteria at mid exponential growth phase in BHI (EC) compared with intracellularly expressed sRNAs at 4 h post-infection in murine macrophages (IC) of rli31, rli33-1, rli50 and rli112.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105390&req=5

Figure 2: Northern blots of sRNA candidates. Validation of RNA-seq data with northern blot analysis of extracellularly expressed sRNAs from bacteria at mid exponential growth phase in BHI (EC) compared with intracellularly expressed sRNAs at 4 h post-infection in murine macrophages (IC) of rli31, rli33-1, rli50 and rli112.
Mentions: The results of the northern blotting, shown in Figure 2, demonstrate signals corresponding to small transcripts from each of the candidates. According to the signals in the northern blot analysis, some of the sRNA candidates were differentially expressed under the tested conditions. rli31 and rli33-1 showed variation in transcript concentration during extra- and intra-cellular growth. Both sRNA candidates exhibited significantly higher transcript numbers during intracellular growth. The northern blot results correlate with read-numbers from transcriptome deep sequencing (Supplementary Table S3). The determined size of rli33-1 by northern blot analysis of ∼140 nt confirms the read length (144 nt) assessed by RNA-seq. rli33-1 showed two bands in the northern blot at ∼200 and 130 nt in length, that probably originate from processing of the larger co-transcript. Both fragments were also detected by transcriptome sequencing excluding the possibility of cross-hybridization (Figure 2). rli50 appears to be longer than predicted by sequencing (306 nt) and has a size of ∼350 nt as determined by northern blot. We observed with an rli112 specific probe strong northern blot signals (∼140 nt) under extra- and intra-cellular conditions indicating high RNA abundance of this new unpublished putative sRNA.Figure 2.

Bottom Line: Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments.A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly.Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, 35392 Giessen, Germany.

ABSTRACT
Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500 nt) isolated from extracellularly growing bacteria and from L. monocytogenes infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here, we report on the discovery of 150 putative regulatory RNAs of which 71 have not been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50* for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNA candidates and the absence of sRNA loci in genomes of naturally occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

Show MeSH
Related in: MedlinePlus